JPH0198955A - Enzyme electrode - Google Patents

Abstract

PURPOSE:To obtain an enzyme electrode which allows mass production, low in cost, has high performance and easy to handle by providing an insulating base plate, >=2 electrodes, insulating protective film and immobilized enzyme film and detecting the concn. of a specific material based on the fluctuation in the enzyme concn. generated by the enzyme reaction in the immobilized enzyme film. CONSTITUTION:This electrode is constituted of the insulating base plate 2, >=2 kinds of the electrodes which respectively have reaction parts 3a, 4a, the insulating protective film 5 which covers and insulates the electrodes except at least the reaction parts 3a, 4a and the immobilized enzyme film 9 which is formed integrally to the base plate surface 2a and covers the electrode reaction parts 3a, 4a. The concn. of the specific chemical material is detected in accordance with the fluctuation in the enzyme concn. generated by the enzyme reaction in this enzyme film 9. Plural sets of the electrodes and the insulating protective film covering the electrodes are collectively provided on a large insulating flat plate and the individual insulating base plate can be formed by dividing this insulating flat plate and, therefore, the mass production of the enzyme electrodes is possible. The production cost of the enzyme electrode is reduced by the improvement in the yield and the reduction of the processing cost.

Description

Detailed Description of the Invention (a) Industrial Application Field This invention relates to an enzyme electrode that detects the concentration of a specific chemical substance based on changes in oxygen concentration caused by an enzyme reaction. , relates to an enzyme electrode that enables improved performance and ease of handling.

(b) Conventional technology Enzyme electrodes are immersed in a liquid to be tested, and as a result of the enzymatic reaction,
By electrochemically measuring changes in the amount of reactants and products, it is possible to detect the concentration of specific chemical substances that are enzyme substrates. , which is equipped with a membrane on which enzymes are immobilized.

Among conventional enzyme electrodes, an enzyme electrode whose cross section is shown in FIG. 7 is known, which is used as an oxygen electrode as a base electrode to measure the concentration of a specific chemical substance based on fluctuations in oxygen concentration.

52 is a working electrode, which is linear (for example, 0.5 to 0.5 in diameter).
1.0 mm) of platinum (pt). This working electrode 52 is insulated by a support member 53. The end face of the support member 53 is ground and polished, and the end face of the working electrode 52 is exposed and serves as a working electrode reaction surface 52a.

54 is a control electrode, which is made of silver (Ag) shaped like a coil spring.
It becomes more. The surface of this control electrode 54 is coated with silver chloride (Ag
A C1 film is formed and serves as a reference electrode reaction surface 54a.

The working electrode 52 and the reference electrode 54 are both housed in a case 55 made of synthetic resin or the like. Lead wires 57 and 57 are connected to the working electrode base end 52b and the control base end 54b, respectively.
They are connected by means such as soldering, and these are hardened with epoxy resin 56. As a result, the working electrode 52 and the reference electrode 54 are coaxially arranged and fixed. At this time, the working electrode reaction surface 52a is connected to the case opening 55.
It is exposed outward from point a and is aligned with the case end surface 55b.

The oxygen permeable membrane 59 is first attached to the case end surface 55b, and the immobilized enzyme membrane 60 is attached thereon in an overlapping manner. For the oxygen permeable membrane 59, a Teflon membrane, a polyethylene membrane, or the like is used. The immobilized enzyme membrane 60 is formed by immobilizing an enzyme on a porous polymer membrane. The oxygen permeable membrane 59 and the immobilized enzyme membrane 60 are connected to the case 5 by the O ring 58.
It is fixed at 5.

An IM or 4M potassium chloride (KCffi) solution, or an IM or 4M potassium hydroxide (KOH) solution is sealed in the cavity 55c inside the case as an electrolyte.

55d is an injection port for this electrolyte.

(c) Problems to be Solved by the Invention The conventional enzyme electrodes mentioned above have been manufactured one by one manually in the manufacturing process, and there has been a problem in that mass production is difficult. This manual work is a series of detailed operations, such as arranging and fixing the working electrode 52 and the reference electrode 54 as shown in FIG. 7, or fixing the working electrode reaction surface 52a and the case end surface 55b. The work of assembling them requires a high degree of skill and careful attention. Further, there was a problem that the loss of materials, especially the loss of electrode materials, was large and the yield was low. Due to this low yield and the above work,
There were also problems in that processing costs were high and the manufacturing cost of the enzyme electrode increased. By the way, this processing cost is 6% of the manufacturing cost.
It accounts for 0% to 80%.

On the other hand, the conventional enzyme electrode 51 described above has the following problems when used. First, there was a problem in that the electrode output fluctuated, noise occurred, and measurement accuracy decreased. This is because grinding and polishing are applied when producing the working electrode reaction surface 52a, but gaps and cracks are created in the support member 53 during this process, and liquid enters into these gaps and cracks. It is something that occurs. Further, the electrode output varies depending on the degree of close contact between the oxygen permeable membrane 59 and the immobilized enzyme membrane 60.

Second, there is a problem in that the electrode output varies between individual enzyme electrodes. This means that the working electrode reaction surface 52a is
This is because, as described above, they are generated manually, and their areas vary.

Thirdly, there is a problem that handling is complicated. This is because, since the electrolyte is sealed inside, there are restrictions on how the electrode can be used, such as having to use the electrode with the case end face 55b facing downward. Furthermore, since the oxygen permeable membrane 59 and the immobilized enzyme membrane 60 are used in a stacked manner, they may be torn during installation, resulting in cumbersome handling.

Another problem was that the response speed was slow because two membranes were stacked on top of each other.

This invention has been made in view of the above, and aims to provide an enzyme electrode that can be mass-produced, is low in cost, has high performance, and is easy to handle.

(d) Means for solving the problems The structure of the enzyme electrode of the present invention will be explained using FIG. 1 (b) and FIG. 2 (6) corresponding to the embodiment. Two or more electrodes 3.4 are formed on the insulating base surface 2a and have reaction parts 3a and 4a having a predetermined area ratio with each other, and the electrodes 3.4 are covered and insulated except for at least these reaction parts 3a and 4a. It comprises an insulating protective film 5 made of photosensitive resin and an immobilized enzyme film 9 that is integrally formed on the insulating base surface 2a and covers the electrode reaction parts 3a and 4a,
The concentration of a specific chemical substance is detected based on the fluctuation in oxygen concentration caused by the enzyme reaction within this immobilized enzyme membrane.

(E) Function The enzyme electrode of the present invention has electrodes 3.4 on the insulating base surface 2a.
However, since multiple sets of electrodes and an insulating protective film covering them are provided on a large insulating flat plate, and this insulating flat plate can be divided into individual insulating bases, the enzyme electrode mass production becomes possible.

In addition, film formation techniques such as vacuum evaporation, sputtering, and printing can be applied to form electrodes, and photolithography technology can be applied to form insulating protective films, but automation of these techniques is well known and easy. Because of this, the manufacturing process of enzyme electrodes can be automated and the processing cost can be reduced.

Furthermore, material loss, especially loss of electrode material, can be reduced.
Yield can be improved. This improvement in yield and reduction in processing costs can reduce the manufacturing cost of enzyme electrodes.

On the other hand, in the enzyme electrode of the present invention, since grinding and polishing of the electrode is not included in the manufacturing process, cracks and gaps do not occur in the insulating base and insulating protective film around the electrode reaction surface. noise is prevented.

Furthermore, the area of the electrode reaction area can be determined with high precision by applying photolithography technology, eliminating variations in output between individual enzyme electrodes.

Furthermore, since the immobilized enzyme membrane is integrally formed on the surface of the insulating base, there is no need to attach two layers of the immobilized enzyme membrane and oxygen permeable membrane as in the past, making handling easier and improving the immobilization. Fluctuations in electrode output caused by differences in the degree of adhesion of enzyme membranes are eliminated.

In addition, since an electrolytic solution is not required, there are no restrictions on the mode of use, and handling becomes easy.

(F) Embodiment An embodiment of the present invention will be described below with reference to FIGS. 1 to 6.

The enzyme electrode according to this example uses glucose oxidase (COD) as an enzyme and is used to measure the glucose concentration in a test liquid such as blood. This example enzyme electrode will be explained while following the manufacturing process.

Figure 1 (a) shows alumina ceramic plate (insulating flat plate) 1
A state in which partition lines 13, . . . , 13 are formed on the surface 12a of 2 is shown. The alumina ceramic plate 12 is
It is later divided to form the insulating substrate 2. For example, it is made of 96% alumina, 50 mm x 50 mm, and 0.4 mm thick.
mm is used. The partition line 13 is a cut formed by laser processing, and its depth is approximately 1/2 of the thickness of the alumina ceramic plate 12 [Fig.
See a)]. Note that the material and shape of the insulating flat plate and the method of forming the partition line are not limited to those described above, and the design can be changed as appropriate.

Working electrodes 3, . . . , 3 are collectively formed in sections 14, . and Figure 2 et al.)]. Each working electrode 3 has a strip shape (for example, lX1
It is a platinum thin film with a thickness of 1 mm and a thickness of 1,500 mm, and is formed by sputtering using a metal mask.

Next, control electrodes 4, . . . , 4 are collectively formed in the sections 14, .
See figure (C)]. Each control electrode 4 has a strip shape (IXIIM,
It is a thin silver film with a thickness of 1,500 mm) and is formed by sputtering or vacuum evaporation. A pair of working electrode 3 and reference electrode 4 will be formed in each section 14 .

A photosensitive polyimide resin (Photovarnish: registered trademark) is applied to the entire surface of the alumina ceramic plate surface 12a to form a photosensitive resin film 15 [FIG. 1(d) and FIG.
See d)]. Of course, this photosensitive resin film 15 is not limited to photosensitive polyimide.

The photosensitive resin film 15 is covered with a photomask (not shown), exposed, developed and rinsed to remove unnecessary parts, and an insulating protective film 5 is formed in each section 14. See Figure (e) and Figure 2 (e)].

The insulating protective film 5 covers and insulates the working electrode 3 and the reference electrode 4.

The insulating protective film 5 has window portions 5aa, 5ab, 5ba, 5.
bb is formed. The window portion 5aa exposes a part of the working electrode 3 to serve as the working electrode reaction surface 3a, and has a size of, for example, 0.2×0.2nvn. Window part 5b
A is for exposing a part of the control electrode 4 to form a control electrode reaction surface 4a, for example, 0.4×5. The size is 0mm. The area ratio of window portion 5aa and window portion 5ba is 1:5.
It is considered to be 0.

On the other hand, the windows 5ab and 5bb expose other parts of the working electrode 3 and the reference electrode 4, respectively, and serve as connection parts 3b and 4b.

Next, the alumina ceramic plate 12 is divided along the partition lines 13 to form individual insulating substrates (insulating bases) 2 [see FIG. 1(f) and FIG. 2(f)]. Each insulation board 2
Lead wires 7.7 are connected to the connecting portions 3b and 4b using conductive adhesive or solder, and sealed with epoxy resin 6. In this state, it is called a base electrode 10.

A silver chloride film 8 is formed on the working electrode reaction surface 3a. This silver chloride film 8 is formed as follows. First, the control electrode reaction surface 4a is placed together with a platinum electrode (not shown).
Immersed in 0.1-1.0M hydrochloric acid solution (HCj2),
Electrolysis may be performed at a current density of 0.1 to 10 mA/crn-2. Although it is not absolutely necessary to form silver chloride IJH on the reference electrode reaction surface 4a, good results can be obtained as an oxygen electrode.

Next, an immobilized enzyme film 9 is formed on the surface of the insulating base 2. First, the base electrode 10 is immersed in a 3% acetylcellulose solution (acetone:cyclohexanone-4:1), and the first acetylcellulose film 9a is formed by dip coating.

Subsequently, enzyme layer 9b is formed. This enzyme layer 9b is
It is formed by dropping a small amount of enzyme solution onto the first acetylcellulose membrane 9a with a micropipette and air drying it. This enzyme solution contains glucose oxidase 2■, 0
．． It is prepared by mixing a solution dissolved in 100 μβ of 1M phosphate buffer (pH 6,0) and a 0.5% glutaraldehyde solution prepared in the same phosphate buffer.

Furthermore, a second acetyl cellulose film 9C is formed on the base electrode 10. This second acetylcellulose film 9c is also formed by dip coating, and is formed by immersing the base electrode 10 in 1 m of 2% acetylcellulose (acetone:ethanol-4=1).

Next, the results of measuring the characteristics of the example enzyme electrode 1 will be explained.

FIG. 3 shows the measurement system 21 used to measure the characteristics. Reference numeral 22 denotes a constant temperature bath, in which a 0.1M phosphate buffer solution 23 adjusted to pH 7.0 is stored at a constant temperature. This phosphate buffer 23 is star 5
24. 25 is a rotor of this star 524.

The lead wire 7 of the enzyme electrode 1 is connected to the electron meter 26
A predetermined applied voltage (10.6 V in this embodiment) is applied. A recorder 27 is connected to the electron meter 26 and records the electrode output (current value) of the enzyme electrode 1.

FIG. 4 shows the results of a test to determine whether the base electrode 10 constituting the enzyme electrode 1 behaves reliably as an oxygen electrode. In this test, sodium hydrosulfide (N az S
z Oa ) solution and phosphate buffer 23 N
a2StO4 concentration 0.4mM and 0.8mM
I have to. The dissolved oxygen level in phosphate buffer 23 is
Although it decreases due to the reducing action of NazS, O, the electrode output also decreases according to the Na25z04 concentration. That is,
It can be confirmed that the base electrode 10 behaves reliably as an oxygen electrode. The horizontal axis in FIG. 4 indicates the elapsed time after injection of the NazS20a solution.

FIG. 5 shows the electrode output (
nA) is plotted. To adjust the phosphate buffer 23 to a predetermined glucose concentration, a predetermined amount of glucose solution (for example, 100 μ!!) is injected. At this time,
FIG. 5 shows that an electrode output is obtained in the immobilized enzyme membrane 9 according to the variation in the oxygen (0□) concentration accompanying this reaction.

An arbitrary glucose concentration can be measured using the line connecting each point in FIG. 5 as a calibration curve.

FIG. 6 shows a modification of the enzyme electrode of this embodiment.

This modification enzyme electrode 31 has a working electrode 33 on one surface 32a of the insulating substrate 32, compared to the enzyme electrode 1 described above.
The difference is that a working electrode 24 is formed on the other surface 32b.

The working electrode 33 is made of a platinum thin film, and is covered and insulated with an insulating protective film 35 made of a photosensitive resin. This insulating protective film 35 is provided with a window 35a, and a part of the working electrode 33 serves as a reaction surface 33a. In addition, the insulating protective film 3
5 is provided with another window (not shown),
The other part of the working electrode 33 is used as a connection part (not shown). A lead wire 37 is connected to this connection portion and sealed with epoxy resin 36.

On the other hand, the reference electrode 34 is made of a thin silver film, and is covered and insulated with an insulating protective film 35''. This insulating protective film 35'' is also provided with a window 35''a, and a part of the reference electrode 34 is is exposed to form a reaction surface 34a. This reaction surface 34
The silver chloride film 38 is formed overlappingly on a.
Same as above.

Another window (not shown) is also provided in the insulating protective film 35', exposing another part of the reference electrode 34 to serve as a connection part (not shown). A lead 37 is also connected to this connection portion and sealed with epoxy resin (not shown).

An immobilized enzyme film 39 is formed on the insulating substrate 32 . This immobilized enzyme membrane 39 is also composed of first and second acetyl cellulose membranes 39a, 39c and an enzyme layer 39b formed by dip coating as before. In this modification, both surfaces 32a and 3 of the insulating substrate 32
Since the working electrode 33 and the control electrode 34 are formed on each of the electrodes 2b, the - layer can be made smaller.

In the above example, glucose oxyguse is immobilized as an enzyme on the immobilized enzyme membrane, but the enzyme is not limited to this, and the design can be changed as appropriate.

(g) As described in detail of the invention, the enzyme electrode of the present invention has the advantage of being mass-producible, and also has the advantage of being able to improve yield and reduce manufacturing costs.

On the other hand, the enzyme electrode of the present invention has the advantage of stable electrode output, low noise, and high precision measurement.

Further, it has the advantage that variations in electrode output between individual enzyme electrodes can be eliminated. Furthermore, it has the advantage that it is easy to handle and there are no restrictions on how it can be used.

[Brief explanation of the drawing]

Figure 1(a), Figure 1(b), Figure 1(C), Figure 1(
d), FIG. 1(e), FIG. 1(f), and FIG. 1(2) are diagrams explaining the manufacturing process of an enzyme electrode according to an embodiment of the present invention, and FIG. 2(a) is a , IIa-Il in FIG. 1(a)
A sectional view of the main part taken along line a, Fig. 2 0)) is similar to Fig. 1 (b
2(C) is a sectional view of the main part taken along line nb-■b in FIG. 1(C),
FIG. 2(d) is a sectional view of the main part taken along the Ird-Ird line in FIG. 1(d), and FIG.
-Ile line sectional view of the main part, FIG. 2(f) is the first
Fig. 2) Cross-sectional view taken along line 11f-Iff in middle
) is a cross-sectional view along the Irg-IIg line in Figure 1 (9), Figure 3 is a diagram explaining the measurement system used to measure the characteristics of the enzyme electrode, and Figure 4 is the structure of the enzyme electrode. FIG. 5 is a diagram showing the relationship between the glucose concentration and electrode output of the enzyme electrode, and FIG. 6(a) is an external perspective view showing a modification of the enzyme electrode. , Figure 6(b)
is a cross-sectional view taken along the vrb-vrb line in FIG. 6(a),
FIG. 7 is a longitudinal sectional view of a conventional enzyme electrode. 1.31: Enzyme electrode, 2.32: Insulating substrate, 3.33:
Working electrode, 4/34: Control electrode, 3a/33a: Working electrode reaction surface, 4a/34a: Control electrode reaction surface, 5/35/35゛: Insulating protective film, 9/39: Immobilized enzyme membrane. Patent Applicant Tateishi Electric Co., Ltd. Representative Patent Attorney Shigeru Nakamura Figure 1 (f) Figure 2 (f) a 2 Q ○ ○ OOO. c'u o co Φ gu O" (V
u) Nine sins? ) (Vu) η Dingsobian “(~IY) IY) Mama Korori”

Claims (1)

[Claims] (1) An insulating base, two or more electrodes formed on the surface of the insulating base and each having a reaction area having a predetermined area ratio to each other, and a photosensitive resin that covers and insulates the electrodes except for at least these reaction areas. an insulating protective film made of An enzyme electrode that detects the concentration of specific chemicals based on